Anatomical and developmental patterns of interleukin-2 gene expression in the mouse: analysis of IL-2 expressing cells and partial characterization of interactions involved in mediating IL-2 gene expression in vivo

Interleukin-2 (IL-2) is an important mediator in the vertebrate immune system. IL-2 is a potent growth factor that mature T lymphocytes use as a proliferation signal and the production of IL-2 is crucial for the clonal expansion of antigen-specific T cells in the primary immune response. IL-2 driven proliferation is dependent on the interaction of the lymphokine with its cognate multichain receptor. IL-2 expression is induced only upon stimulation and transcriptional activation of the IL-2 gene relies extensively on the coordinate interaction of numerous inducible and constitutive trans-acting factors. Over the past several years, thousands of papers have been published regarding molecular and cellular aspects of IL-2 gene expression and IL-2 function. The vast majority of these reports describe work that has been carried out in vitro. However, considerably less is known about control of IL-2 gene expression and IL-2 function in vivo.

To gain new insight into the regulation of IL-2 gene expression in vivo, anatomical and developmental patterns of IL-2 gene expression in the mouse were established by employing in situ hybridization and immunohistochemical staining methodologies to tissue sections generated from normal mice and mutant animals in which T -cell development was perturbed. Results from these studies revealed several interesting aspects of IL-2 gene expression, such as (1) induction of IL-2 gene expression and protein synthesis in the thymus, the primary site of T-cell development in the body, (2) cell-type specificity of IL-2 gene expression in vivo, (3) participation of IL-2 in the extrathymic expansion of mature T cells in particular tissues, independent of an acute immune response to foreign antigen, (4) involvement of IL-2 in maintaining immunologic balance in the mucosal immune system, and (5) potential function of IL-2 in early events associated with hematopoiesis.

Extensive analysis of IL-2 mRNA accumulation and protein production in the murine thymus at various stages of development established the existence of two classes of intrathymic IL-2 producing cells. One class of intrathymic IL-2 producers was found exclusively in the fetal thymus. Cells belonging to this subset were restricted to the outermost region of the thymus. IL-2 expression in the fetal thymus was highly transient; a dramatic peak ofiL-2 mRNA accumulation was identified at day 14.5 of gestation and maximal IL-2 protein production was observed 12 hours later, after which both IL-2 mRNA and protein levels rapidly decreased. Significantly, the presence of IL-2 expressing cells in the day 14-15 fetal thymus was not contingent on the generation of T-cell receptor (TcR) positive cells. The second class of IL-2 producing cells was also detectable in the fetal thymus (cells found in this class represented a minority subset of IL-2 producers in the fetal thymus) but persist in the thymus during later stages of development and after birth. Intrathymic IL-2 producers in postnatal animals were located in the subcapsular region and cortex, indicating that these cells reside in the same areas where immature T cells are consigned. The frequency of IL-2 expressing cells in the postnatal thymus was extremely low, indicating that induction of IL-2 expression and protein synthesis are indicative of a rare activation event. Unlike the fetal class of intrathymic IL-2 producers, the presence of IL-2 producing cells in the postnatal thymus was dependent on to the generation of TcR+ cells. Subsequent examination of intrathymic IL-2 production in mutant postnatal mice unable to produce either αβ or γδ T cells showed that postnatal IL-2 producers in the thymus belong to both αβ and γδ lineages. Additionally, further studies indicated that IL-2 synthesis by immature αβ -T cells depends on the expression of bonafide TcR αβ-heterodimers. Taken altogether, IL-2 production in the postnatal thymus relies on the generation of αβ or γδ-TcR^+ cells and induction of IL-2 protein synthesis can be linked to an activation event mediated via the TcR.

With regard to tissue specificity of IL-2 gene expression in vivo, analysis of whole body sections obtained from normal neonatal mouse pups by in situ hybridization demonstrated that IL-2 mRNA^+ cells were found in both lymphoid and nonlymphoid tissues with which T cells are associated, such as the thymus (as described above), dermis and gut. Tissues devoid of IL-2 mRNA^+ cells included brain, heart, lung, liver, stomach, spine, spinal cord, kidney, and bladder. Additional analysis of isolated tissues taken from older animals revealed that IL-2 expression was undetectable in bone marrow and in nonactivated spleen and lymph nodes. Thus, it appears that extrathymic IL-2 expressing cells in nonimmunologically challenged animals are relegated to particular epidermal and epithelial tissues in which characterized subsets of T cells reside and thatinduction of IL-2 gene expression associated with these tissues may be a result of T-cell activation therein.

Based on the neonatal in situ hybridization results, a detailed investigation into possible induction of IL-2 expression resulting in IL-2 protein synthesis in the skin and gut revealed that IL-2 expression is induced in the epidermis and intestine and IL-2 protein is available to drive cell proliferation of resident cells and/or participate in immune function in these tissues. Pertaining to IL-2 expression in the skin, maximal IL-2 mRNA accumulation and protein production were observed when resident Vγ_3^+ T-cell populations were expanding. At this age, both IL-2 mRNA^+ cells and IL-2 protein production were intimately associated with hair follicles. Likewise, at this age a significant number of CD3ε^+ cells were also found in association with follicles. The colocalization of IL-2 expression and CD3ε^+ cells suggests that IL-2 expression is induced when T cells are in contact with hair follicles. In contrast, neither IL-2 mRNA nor IL-2 protein were readily detected once T-cell density in the skin reached steady-state proportions. At this point, T cells were no longer found associated with hair follicles but were evenly distributed throughout the epidermis. In addition, IL-2 expression in the skin was contingent upon the presence of mature T cells therein and induction of IL-2 protein synthesis in the skin did not depend on the expression of a specific TcR on resident T cells. These newly disclosed properties of IL-2 expression in the skin indicate that IL-2 may play an additional role in controlling mature T-cell proliferation by participating in the extrathymic expansion of T cells, particularly those associated with the epidermis.

Finally, regarding IL-2 expression and protein synthesis in the gut, IL-2 producing cells were found associated with the lamina propria of neonatal animals and gut-associated IL-2 production persisted throughout life. In older animals, the frequency of IL-2 producing cells in the small intestine was not identical to that in the large intestine and this difference may reflect regional specialization of the mucosal immune system in response to enteric antigen. Similar to other instances of IL-2 gene expression in vivo, a failure to generate mature T cells also led to an abrogation of IL-2 protein production in the gut. The presence of IL-2 producing cells in the neonatal gut suggested that these cells may be generated during fetal development. Examination of the fetal gut to determine the distribution of IL-2 producing cells therein indicated that there was a tenfold increase in the number of gut-associated IL-2 producers at day 20 of gestation compared to that observed four days earlier and there was little difference between the frequency of IL-2 producing cells in prenatal versus neonatal gut. The origin of these fetally-derived IL-2 producing cells is unclear. Prior to the immigration of IL-2 inducible cells to the fetal gut and/or induction of IL-2 expression therein, IL-2 protein was observed in the fetal liver and fetal omentum, as well as the fetal thymus. Considering that induction of IL-2 protein synthesis may be an indication of future functional capability, detection of IL-2 producing cells in the fetal liver and fetal omentum raises the possibility that IL-2 producing cells in the fetal gut may be extrathymic in origin and IL-2 producing cells in these fetal tissues may not belong solely to the T lineage. Overall, these results provide increased understanding of the nature of IL-2 producing cells in the gut and how the absence of IL-2 production therein and in fetal hematopoietic tissues can result in the acute pathology observed in IL-2 deficient animals.

I. Impact spallation experiments: fracture patterns and spall velocities. II. Craters in carbonate rocks: an electron paramagnetic resonance analysis of shock damage

This work is divided into two independent papers.

PAPER 1.

Spall velocities were measured for nine experimental impacts into San Marcos gabbro targets. Impact velocities ranged from 1 to 6.5 km/sec. Projectiles were iron, aluminum, lead, and basalt of varying sizes. The projectile masses ranged from a 4 g lead bullet to a 0.04 g aluminum sphere. The velocities of fragments were measured from high-speed films taken of the events. The maximum spall velocity observed was 30 m/sec, or 0.56 percent of the 5.4 km/sec impact velocity. The measured velocities were compared to the spall velocities predicted by the spallation model of Melosh (1984). The compatibility between the spallation model for large planetary impacts and the results of these small scale experiments are considered in detail.

The targets were also bisected to observe the pattern of internal fractures. A series of fractures were observed, whose location coincided with the boundary between rock subjected to the peak shock compression and a theoretical "near surface zone" predicted by the spallation model. Thus, between this boundary and the free surface, the target material should receive reduced levels of compressive stress as compared to the more highly shocked region below.

PAPER 2.

Carbonate samples from the nuclear explosion crater, OAK, and a terrestrial impact crater, Meteor Crater, were analyzed for shock damage using electron para- magnetic resonance, EPR. The first series of samples for OAK Crater were obtained from six boreholes within the crater, and the second series were ejecta samples recovered from the crater floor. The degree of shock damage in the carbonate material was assessed by comparing the sample spectra to spectra of Solenhofen limestone, which had been shocked to known pressures.

The results of the OAK borehole analysis have identified a thin zone of highly shocked carbonate material underneath the crater floor. This zone has a maximum depth of approximately 200 ft below sea floor at the ground zero borehole and decreases in depth towards the crater rim. A layer of highly shocked material is also found on the surface in the vicinity of the reference bolehole, located outside the crater. This material could represent a fallout layer. The ejecta samples have experienced a range of shock pressures.

It was also demonstrated that the EPR technique is feasible for the study of terrestrial impact craters formed in carbonate bedrock. The results for the Meteor Crater analysis suggest a slight degree of shock damage present in the β member of the Kaibab Formation exposed in the crater walls.

Applications of frequency modulation interference cancellers to multiaccess communications systems

Cancellation of interfering frequency-modulated (FM) signals is investigated with emphasis towards applications on the cellular telephone channel as an important example of a multiple access communications system. In order to fairly evaluate analog FM multiaccess systems with respect to more complex digital multiaccess systems, a serious attempt to mitigate interference in the FM systems must be made. Information-theoretic results in the field of interference channels are shown to motivate the estimation and subtraction of undesired interfering signals. This thesis briefly examines the relative optimality of the current FM techniques in known interference channels, before pursuing the estimation and subtracting of interfering FM signals.

The capture-effect phenomenon of FM reception is exploited to produce simple interference-cancelling receivers with a cross-coupled topology. The use of phase-locked loop receivers cross-coupled with amplitude-tracking loops to estimate the FM signals is explored. The theory and function of these cross-coupled phase-locked loop (CCPLL) interference cancellers are examined. New interference cancellers inspired by optimal estimation and the CCPLL topology are developed, resulting in simpler receivers than those in prior art. Signal acquisition and capture effects in these complex dynamical systems are explained using the relationship of the dynamical systems to adaptive noise cancellers.

FM interference-cancelling receivers are considered for increasing the frequency reuse in a cellular telephone system. Interference mitigation in the cellular environment is seen to require tracking of the desired signal during time intervals when it is not the strongest signal present. Use of interference cancelling in conjunction with dynamic frequency-allocation algorithms is viewed as a way of improving spectrum efficiency. Performance of interference cancellers indicates possibilities for greatly increased frequency reuse. The economics of receiver improvements in the cellular system is considered, including both the mobile subscriber equipment and the provider's tower (base station) equipment.

The thesis is divided into four major parts and a summary: the introduction, motivations for the use of interference cancellation, examination of the CCPLL interference canceller, and applications to the cellular channel. The parts are dependent on each other and are meant to be read as a whole.

Investigations of noise and of quantum interference in proximity effect bridges

This work reports investigations upon weakly superconducting proximity effect bridges. These bridges, which exhibit the Josephson effects, are produced by bisecting a superconductor with a short (<1µ) region of material whose superconducting transition temperature is below that of the adjacent superconductors. These bridges are fabricated from layered refractory metal thin films whose transition temperature will depend upon the thickness ratio of the materials involved. The thickness ratio is changed in the area of the bridge to lower its transition temperature. This is done through novel photolithographic techniques described in the text, Chapter 2.

If two such proximity effect bridges are connected in parallel, they form a quantum interferometer. The maximum zero voltage current through this circuit is periodically modulated by the magnetic flux through the circuit. At a constant bias current, the modulation of the critical current produces a modulation in the dc voltage across the bridge. This change in dc voltage has been found to be the result of a change in the internal dissipation in the device. A simple model using lumped circuit theory and treating the bridges as quantum oscillators of frequency ω = 2eV/h, where V is the time average voltage across the device, has been found to adequately describe the observed voltage modulation.

The quantum interferometers have been converted to a galvanometer through the inclusion of an integral thin film current path which couples magnetic flux through the interferometer. Thus a change in signal current produces a change in the voltage across the interferometer at a constant bias current. This work is described in Chapter 3 of the text.

The sensitivity of any device incorporating proximity effect bridges will ultimately be determined by the fluctuations in their electrical parameters. He have measured the spectral power density of the voltage fluctuations in proximity effect bridges using a room temperature electronics and a liquid helium temperature transformer to match the very low (~ 0.1 Ω) impedances characteristic of these devices.

We find the voltage noise to agree quite well with that predicted by phonon noise in the normal conduction through the bridge plus a contribution from the superconducting pair current through the bridge which is proportional to the ratios of this current to the time average voltage across the bridge. The total voltage fluctuations are given by <V^2(f ) > = 4kTR^2_d I/V where R_d is the dynamic resistance, I the total current, and V the voltage across the bridge . An additional noise source appears with a strong 1/f^(n) dependence , 1.5 < n < 2, if the bridges are fabricated upon a glass substrate. This excess noise, attributed to thermodynamic temperature fluctuations in the volume of the bridge, increases dramatically on a glass substrate due to the greatly diminished thermal diffusivity of the glass as compared to sapphire.

Radiation from a short electric dipole antenna in a hot uniaxial plasma

The effects of electron temperature on the radiation fields and the resistance of a short dipole antenna embedded in a uniaxial plasma have been studied. It is found that for ω < ω_p the antenna excites two waves, a slow wave and a fast wave. These waves propagate only within a cone whose axis is parallel to the biasing magnetostatic field B_o and whose semicone angle is slightly less than sin ^(-1) (ω/ω_p). In the case of ω > ω_p the antenna excites two separate modes of radiation. One of the modes is the electromagnetic mode, while the other mode is of hot plasma origin. A characteristic interference structure is noted in the angular distribution of the field. The far fields are evaluated by asymptotic methods, while the near fields are calculated numerically. The effects of antenna length ℓ, electron thermal speed, collisional and Landau damping on the near field patterns have been studied.

The input and the radiation resistances are calculated and are shown to remain finite for nonzero electron thermal velocities. The effect of Landau damping and the antenna length on the input and radiation resistances has been considered.

The radiation condition for solving Maxwell's equations is discussed and the phase and group velocities for propagation given. It is found that for ω < ω_p in the radial direction (cylindrical coordinates) the power flow is in the opposite direction to that of the phase propagation. For ω > ω_p the hot plasma mode has similar characteristics.

Quantum Interference and Entanglement of Surface Plasmons

Surface plasma waves arise from the collective oscillations of billions of electrons at the surface of a metal in unison. The simplest way to quantize these waves is by direct analogy to electromagnetic fields in free space, with the surface plasmon, the quantum of the surface plasma wave, playing the same role as the photon. It follows that surface plasmons should exhibit all of the same quantum phenomena that photons do, including quantum interference and entanglement.

Unlike photons, however, surface plasmons suffer strong losses that arise from the scattering of free electrons from other electrons, phonons, and surfaces. Under some circumstances, these interactions might also cause “pure dephasing,” which entails a loss of coherence without absorption. Quantum descriptions of plasmons usually do not account for these effects explicitly, and sometimes ignore them altogether. In light of this extra microscopic complexity, it is necessary for experiments to test quantum models of surface plasmons.

In this thesis, I describe two such tests that my collaborators and I performed. The first was a plasmonic version of the Hong-Ou-Mandel experiment, in which we observed two-particle quantum interference between plasmons with a visibility of 93 ± 1%. This measurement confirms that surface plasmons faithfully reproduce this effect with the same visibility and mutual coherence time, to within measurement error, as in the photonic case.

The second experiment demonstrated path entanglement between surface plasmons with a visibility of 95 ± 2%, confirming that a path-entangled state can indeed survive without measurable decoherence. This measurement suggests that elastic scattering mechanisms of the type that might cause pure dephasing must have been weak enough not to significantly perturb the state of the metal under the experimental conditions we investigated.

These two experiments add quantum interference and path entanglement to a growing list of quantum phenomena that surface plasmons appear to exhibit just as clearly as photons, confirming the predictions of the simplest quantum models.

Nonclassical excitation and quantum interference in a three level atom

Non-classical properties and quantum interference (QI) in two-photon excitation of a three level atom (|1〉), |2〉, |3〉) in a ladder configuration, illuminated by multiple fields in non-classical (squeezed) and/or classical (coherent) states, is studied. Fundamentally new effects associated with quantum correlations in the squeezed fields and QI due to multiple excitation pathways have been observed. Theoretical studies and extrapolations of these findings have revealed possible applications which are far beyond any current capabilities, including ultrafast nonlinear mixing, ultrafast homodyne detection and frequency metrology. The atom used throughout the experiments was Cesium, which was magneto-optically trapped in a vapor cell to produce a Doppler-free sample. For the first part of the work the |1〉 → |2〉 → |3〉 transition (corresponding to the 6S_1/2F = 4 → 6P_3/2F' = 5 → 6D_5/2F" = 6 transition) was excited by using the quantum-correlated signal (Ɛ_s) and idler (Ɛ_i) output fields of a subthreshold non-degenerate optical parametric oscillator, which was tuned so that the signal and idler fields were resonant with the |1〉 → |2〉 and |2〉 → |3〉 transitions, respectively. In contrast to excitation with classical fields for which the excitation rate as a function of intensity has always an exponent greater than or equal to two, excitation with squeezed-fields has been theoretically predicted to have an exponent that approaches unity for small enough intensities. This was verified experimentally by probing the exponent down to a slope of 1.3, demonstrating for the first time a purely non-classical effect associated with the interaction of squeezed fields and atoms. In the second part excitation of the two-photon transition by three phase coherent fields Ɛ₁ , Ɛ₂ and Ɛ₀, resonant with the dipole |1〉 → |2〉 and |2〉 → |3〉 and quadrupole |1〉 → |3〉 transitions, respectively, is studied. QI in the excited state population is observed due to two alternative excitation pathways. This is equivalent to nonlinear mixing of the three excitation fields by the atom. Realizing that in the experiment the three fields are spaced in frequency over a range of 25 THz, and extending this scheme to other energy triplets and atoms, leads to the discovery that ranges up to 100's of THz can be bridged in a single mixing step. Motivated by these results, a master equation model has been developed for the system and its properties have been extensively studied.

Firing Patterns of Cerebellar Purkinje Cells During Locomotion and Sleep

The cerebellum is a major supraspinal center involved in the coordination of movement. The principal neurons of the cerebellar cortex, Purkinje cells, receive excitatory synaptic input from two sources: the parallel and climbing fibers. These pathways have markedly different effects: the parallel fibers control the rate of simple sodium spikes, while the climbing fibers induce characteristic complex spike bursts, which are accompanied by dendritic calcium transients and play a key role in regulating synaptic plasticity. While many studies using a variety of species, behaviors, and cerebellar regions have documented modulation in Purkinje cell activity during movement, few have attempted to record from these neurons in unrestrained rodents. In this dissertation, we use chronic, multi-tetrode recording in freely-behaving rats to study simple and complex spike firing patterns during locomotion and sleep. Purkinje cells discharge rhythmically during stepping, but this activity is highly variable across steps. We show that behavioral variables systematically influence the step-locked firing rate in a step-phase-dependent way, revealing a functional clustering of Purkinje cells. Furthermore, we find a pronounced disassociation between patterns of variability driven by the parallel and climbing fibers, as well as functional differences between cerebellar lobules. These results suggest that Purkinje cell activity not only represents step phase within each cycle, but is also shaped by behavior across steps, facilitating control of movement under dynamic conditions. During sleep, we observe an attenuation of both simple and complex spiking, relative to awake behavior. Although firing rates during slow wave sleep (SWS) and rapid eye movement sleep (REM) are similar, simple spike activity is highly regular in SWS, while REM is characterized by phasic increases and pauses in simple spiking. This phasic activity in REM is associated with pontine waves, which propagate into the cerebellar cortex and modulate both simple and complex spiking. Such a temporal coincidence between parallel and climbing fiber activity is known to drive plasticity at parallel fiber synapses; consequently, pontocerebellar waves may provide a mechanism for tuning synaptic weights in the cerebellum during active sleep.